Phosphoinositide-3 kinases (PI3K) belong to a family of lipid kinases characterized by their ability to phosphorylate the 3'OH group of phosphoinositides and generate second messengers such as phosphatidylinositol-3,4,5-triphosphate that are critical for the regulation of immune responses. Among the various subclasses of PI3K, PI3K? and ? have received much attention for their restricted expression in leukocytes and their regulatory role in immune cells and inflammation. Nevertheless, despite an accumulating body of information on the role of PI3K? and ? in inflammation, the mechanisms by which the PI3K? and ? signaling pathways function in alloreactive T effector/regulatory cells remain unexplored. Over the past several years, we have generated a substantial amount of data indicating that PI3K? inhibition markedly reduces both acute and chronic heart allograft rejection. PI3K? inhibition results in selective suppression of T effector cells (Teff) while augmenting T regulatory cells (Treg). Similarly, PI3K? inhibition suppresses alloreactive T cells and increases heart allograft survival in a MHC-mismatched heart transplant model. However, our PI3K? inhibition data indicate that PI3K? has a counter-regulatory function as opposed to PI3K? signaling, shown by down-regulation of Tregs. As Teff and Treg require distinct metabolic machinery to meet their energy demands, our data indicate that the PI3K pathway plays a key role in determining the fate of Teff and Treg by critically regulating their metabolic programming. Our overall objective is to decipher the relative contribution of the PI3K? and ? subclasses in T cell-dependent alloimmunity and use the new information to support the discovery of therapeutics that have high potential to enhance immunoregulation in alloimmunity. We hypothesize that PI3K? and PI3K? activation results in the expansion of alloreactive effector T cells; however PI3K? and PI3K? have counter-regulatory effects in Treg homeostasis. In Aim 1, we define the mechanisms by which PI3K? and ? inhibition down- regulates alloantigen-specific Teff. In Aim 2, we will determine the differential impact of PI3K? and ? inhibition on alloantige-specific Treg. These studies will deploy murine heart transplant models in CD4 and CD8 alloantigen-specific hosts (ABM-tg and 2C-tg mice) to elucidate the fate of alloreactive T cells in vivo. These models accurately assess the effects of PI3K? and ? inhibition on T cell activation, mechanisms of anergy, Th/Tc differentiation, and T cell apoptosis. In Aim 3, we will evaluate the differential impact of PI3K? and ? on Treg and Teff metabolic programming in vitro. Alloimmune responses are critically determined by the balance of alloreactive T cells and regulatory T cells. Identifying novel regulatory pathways which control this balance has a significant impact on the design of future immunomodulatory therapies in transplantation. These studies will provide fundamental information on the role of PI3K? and ? in controlling the balance of Teff/ Treg, critical information required for the design of innovative, significant, effective and safer strategies to reduce alloimmunity and promote graft acceptance.